PROJECT SUMMARY/ABSTRACT Immune checkpoint blockade is a major breakthrough in cancer immunotherapy that promotes the immune system?s response against tumors. Unfortunately, this immunotherapy is ineffective in the majority of cancer patients, posing a major clinical problem. The microbial organisms in the gut have a profound influence in educating the immune system as well as dysregulating the immune system to undermine immunotherapy success. These gut microbes are manipulatable through microbial transplants or prebiotics/probiotics and thus offers an practicable way of improving efficacy. This proposal will investigate the mechanisms by which the gut microbiome influences the human immune system in the context of immune checkpoint inhibitors (ICIs) to ultimately improve cancer treatment. Recent advances have shown that the gut microbiome plays an influential role in ICI efficacy, but the underlying mechanisms are unknown. I hypothesize that it is the microbial metabolites from particular gut commensals that directly influence immune cell response to ICIs and thus causes the differences seen in ICI clinical efficacy. From our preliminary experiments, we have identified four commensal gut bacteria that produce products to directly stimulate human immune cell production of interferon-?, a central orchestrator of antitumor immune response. To further investigate how these bacteria alter the immune system to influence ICI efficacy, I propose two aims. In Aim 1, I will identify the microbial metabolite producing gene responsible for inducing this immune effect by creating a Fosmid library of the microbial genome of each of these four bacteria. These clones will be tested in an in-vitro immune assay to identify which part of the microbial genome specifically influences interferon production. In Aim 2, I will demonstrate the in-vivo effects of these bacteria by transplanting these four commensal gut bacteria in a model by which healthy mice with a humanized immune system develop autoimmune symptoms when treated with an ICI. By administering these bacteria and their knockout mutants of the immunomodulatory metabolite gene in these humanized mouse, we are able to assess the severity of autoimmune response as a readout of ICI effectiveness. The goal of this project is to find a bacterium that increases ICI efficacy and identify what gene in the bacterium is producing the immunomodulatory metabolite. By advancing our understanding of microbiome?immune system interactions, findings from this project are anticipated to stimulate improvements in clinical ICI treatment protocols. For example, manipulation of a cancer patient?s gut microbial composition prior to immunotherapy could be a highly advantageous strategy to increase ICI efficacy.